title: A homogeneous single-label quenching resonance energy transfer assay for a δ-opioid receptor-ligand using intact cells.
abstract: This study, a homogeneous assay system for delta opioid receptor binding ligands has been developed using Quenching Resonance Energy Transfer (QRET). The QRET system allows receptor-ligand binding assays on intact cells using a single-label approach and a nonspecific quenching mechanism. Binding of antagonists or agonists to the receptor can be defined using a europium(III) labeled ligand. In the presence of the unlabeled ligand the labeled ligand is displaced and remains in solution. The non-bound labeled ligand is not protected by the target receptor, and the luminescence signal is quenched. For this objective, a Eu(III) labeled peptide molecule with three different linkers (AX0, AX1 and AX2) was designed. Peptides were evaluated using the homogeneous QRET technique, radioligand binding assays and the heterogeneous time-resolved luminescence (TRL) technique. Using the Eu-AX0 peptide and the QRET method, a panel of opioid compounds (naltrexone, naltrindole, SCN-80, DPDPE and DAMGO) was tested to prove the assay performance. The signal-to-background ratio for the tested opioid ligand ranged from 3.3 to 12.0. The QRET method showed prominent performance also in high DMSO concentrations. QRET is a homogenous and a non-radioactive detection system for screening and this is the first attempt to utilize peptide ligands in the QRET concept.
authors.affiliation: Laboratory of Biophysics, Department of Cell Biology and Anatomy, Institute of Biomedicine, University of Turku, Tykistökatu 6 A 5th floor, FI-20520 Turku, Finland. khkopr@utu.fi
authors.forename: Kari
authors.initials: K
authors.lastname: Kopra
authors.lastname: Shweta
authors.forename: Eija
authors.initials: E
authors.lastname: Martikkala
authors.forename: Pekka
authors.initials: P
authors.lastname: Hänninen
authors.forename: Ulla
authors.initials: U
authors.lastname: Petäjä-Repo
authors.forename: Harri
authors.initials: H
authors.lastname: Härmä
mesh_terms: D002452:Cell Count; D004121:Dimethyl Sulfoxide / Q000737:chemistry; D005063:Europium / Q000737:chemistry; D031541:Fluorescence Resonance Energy Transfer; D057809:HEK293 Cells; D006801:Humans; D008024:Ligands; D008958:Models, Molecular; D009842:Oligopeptides / Q000737:chemistry / Q000378:metabolism; D011487:Protein Conformation; D017465:Receptors, Opioid, delta / Q000737:chemistry / Q000378:metabolism*; D013194:Staining and Labeling
chemical_list: D008024:Ligands; D009842:Oligopeptides; D017465:Receptors, Opioid, delta; D005063:Europium; D004121:Dimethyl Sulfoxide